Project 3: Demethylation of HPV-associated head and neck cancer to trigger APOBEC synthetic lethality and enhance immune response

项目3：HPV相关头颈癌去甲基化触发APOBEC合成致死性并增强免疫反应

• 批准号: 10441511
• 负责人: Karen S. Anderson
• 金额: $ 43.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-22 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10441511
• 关键词: AXIN1 protein; Aftercare; Azacitidine; Bioinformatics; Biological Assay; Biometry; Cancer Etiology; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Cessation of life; Characteristics; Chromatin; Clinical Data; Clinical Trials; Clonal Expansion; Clustered Regularly Interspaced Short Palindromic Repeats; Cytidine Deaminase; DNA; DNA Damage; Data; Dependence; Diagnosis; Disease; Dose; Enrollment; Epidemic; Flow Cytometry; GAGE; Genes; Genetic Transcription; Genomics; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Health; Human Papilloma Virus Vaccine; Human Papilloma Virus-Related Malignant Neoplasm; Human Papillomavirus; Hypermethylation; Immune; Immune checkpoint inhibitor; Immune response; Immunofluorescence Immunologic; In Vitro; Incidence; Individual; Infiltration; Innate Immune Response; Interferon Type I; Interferons; Lymphocyte; Malignant Epithelial Cell; Malignant Neoplasms; Measures; Mediating; Methylation; Molecular; Morbidity - disease rate; Mutagenesis; Mutate; Mutation; Neck; Nivolumab; Outcome; Patients; Prognosis; Recurrence; Recurrent disease; Role; Sampling; Signal Pathway; Signal Transduction; Specimen; Squamous cell carcinoma; Stains; Supporting Cell; T-Lymphocyte; Testing; Testis; The Cancer Genome Atlas; Toxic effect; Tumor-infiltrating immune cells; Xenograft procedure; apolipoprotein B mRNA editing enzyme; base; cancer cell; cancer testis antigen; cell killing; cervical and uterine cancer; cytotoxic; cytotoxicity; demethylation; effective therapy; efficacy testing; immune activation; in vitro Assay; knock-down; neoantigens; novel; novel therapeutics; overexpression; patient population; patient prognosis; polypeptide; pre-clinical; prevent; programmed cell death protein 1; response; sensor; side effect; therapeutically effective; therapy resistant; transcriptome sequencing; tumor; tumor microenvironment; tumor xenograft

SUMMARY Human papillomavirus (HPV)-associated neck squamous cell carcinoma (HNSCC) represents an increasing proportion of HNSCC. The incidence of HPV+ HNSCC has dramatically increased over the last 2 decades and in 2012 surpassed uterine cervical cancer as the most common HPV-related malignancy in the U.S. Despite the HPV vaccine, it is estimated that the “epidemic” of HNSCC caused by HPV will not diminish until 2060. HPV+ HNSCCs occur in younger individuals and prognosis for patients with these tumors is better compared to patients with classical HNSCC; however, ~25% of patients recur with few effective therapeutic options. Based on observed hypermethylation of HPV+ HNSCC from TCGA, and understanding that HPV uses hypermethylation to impede the innate immune response, effects of the demethylating agent, 5-azacytidine (5- azaC), were tested on HPV+ HNSCC. We found that HPV+ HNSCC cells in culture and xenografts are sensitive to 5-azaC, and that 5-azaC caused double strand breaks (DSB) that were not observed after 5-azaC therapy in HPV-negative HNSCC, even with much higher doses. We found that following 5-azaC therapy, APOlipoprotein B mRNA-Editing enzyme Catalytic polypeptide 3B (APOBEC3B) was associated with chromatin in HPV+ HNSCC, but not HPV-negative cells. CRISPR knockdown of A3B prevented DSB and protected cells from 5-azaC-induced death. Despite being required for DSBs and cellular toxicity caused by 5- azaC, A3B was also required for clonogenic survival of untreated HPV+ HNSCC. These data showing that A3B is required for survival of HPV+ HNSCC cells, but that following demethylation A3B mediates toxicity and DSB. In addition, 5-azaC therapy increased type I interferon signaling as measured by increased expression of interferon-stimulated genes. These exciting pre-clinical data led to a window trial of 5days of 5-azaC. Analysis of tumor specimens confirmed in vitro data showing that 5-azaC resulted in cellular toxicity. Immunofluorescent staining of an HPV+ patient tumors pre- and post-5-azaC showed a marked increase in tumor-associated lymphocytes, possibly driven through activation of type I interferon combined with increased expression of neoantigens. In this YHN-SPORE project, we hypothesize 5-azaC therapy will enhance response to nivolumab (Nivo) through its ability to cause cell death, increase neoantigen expression, increase A3B-driven mutational load, and enhance T cell infiltration through increased type I interferon signaling. These hypotheses will be tested using established and novel in vitro assays, as well as through examination of pre- and post-therapy tumor specimens from a 3-armed clinical trial. In Aim 1, tumor specimens from the SPORE window trial will be analyzed to determine effects of 5-azaC, Nivo, or the combination on cell death, cell proliferation, immune infiltration and immune activation. Aim 2 will employ standard and novel assays to explore the role of A3B in cellular toxicity exposed by 5-azaC therapy. In Aim 3, we will determine effects of 5-azaC on activators of immune recognition and response in the presence or absence of Nivo.

概括 人乳头瘤病毒 (HPV) 相关的颈部鳞状细胞癌 (HNSCC) 代表着越来越多的人 HNSCC 的比例。过去 20 年来，HPV+ HNSCC 的发病率急剧增加 2012年超过宫颈癌成为美国最常见的HPV相关恶性肿瘤 HPV疫苗问世后，预计到2060年，HPV引起的头颈部鳞状细胞癌的“流行”不会减少。 HPV+ HNSCC 发生在较年轻的个体中，并且这些肿瘤患者的预后比较好 典型 HNSCC 患者；然而，约 25% 的患者在几乎没有有效的治疗选择的情况下会复发。 基于从 TCGA 观察到的 HPV+ HNSCC 的高甲基化，并了解 HPV 使用 过度甲基化会阻碍先天免疫反应，去甲基化剂 5-氮杂胞苷 (5- azaC)，在 HPV+ HNSCC 上进行了测试。我们发现培养物和异种移植物中的 HPV+ HNSCC 细胞是 对 5-azaC 敏感，并且 5-azaC 会引起双链断裂 (DSB)，而在 5-azaC 后未观察到 HPV 阴性 HNSCC 的治疗，即使剂量要高得多。我们发现在 5-azaC 治疗后， APO 脂蛋白 B mRNA 编辑酶催化多肽 3B (APOBEC3B) 与 HPV+ HNSCC 中存在染色质，但 HPV 阴性细胞中不存在染色质。 A3B 的 CRISPR 敲低可阻止 DSB 并 保护细胞免受 5-azaC 诱导的死亡。尽管需要 DSB 和 5- 引起的细胞毒性 azaC、A3B 也是未经治疗的 HPV+ HNSCC 克隆存活所必需的。这些数据表明A3B A3B 是 HPV+ HNSCC 细胞存活所必需的，但去甲基化后的 A3B 会介导毒性和 DSB。 此外，通过增加 I 型干扰素的表达来测量，5-azaC 治疗增加了 I 型干扰素信号传导。 干扰素刺激的基因。这些令人兴奋的临床前数据导致了 5-azaC 为期 5 天的窗口试验。分析 肿瘤标本证实了体外数据显示 5-azaC 会导致细胞毒性。免疫荧光 对 HPV+ 患者肿瘤在 5-azaC 治疗前和治疗后的染色显示，肿瘤相关细胞因子显着增加。 淋巴细胞，可能是通过 I 型干扰素的激活以及 I 型干扰素表达的增加来驱动的 新抗原。在这个 YHN-SPORE 项目中，我们假设 5-azaC 疗法将增强对纳武单抗的反应 (Nivo) 通过其导致细胞死亡、增加新抗原表达、增加 A3B 驱动的突变的能力 负荷，并通过增加 I 型干扰素信号传导增强 T 细胞浸润。这些假设将 使用已建立的和新颖的体外测定以及通过治疗前和治疗后的检查进行测试 来自三组临床试验的肿瘤标本。在目标 1 中，来自 SPORE 窗口试验的肿瘤标本将被 分析以确定 5-azaC、Nivo 或组合对细胞死亡、细胞增殖、免疫的影响 渗透和免疫激活。目标 2 将采用标准和新颖的检测方法来探索 A3B 在 5-azaC 疗法暴露的细胞毒性。在目标 3 中，我们将确定 5-azaC 对激活剂的影响 在存在或不存在 Nivo 的情况下的免疫识别和反应。